The present invention relates to a method of analyzing the distribution of a reagent, following its reaction, between particles and liquid in a suspension and, more particularly, wherein said analysis does not require the separation of the particles from the liquid in order to measure the distribution of the reagent.
As used hereinabove and hereinafter, the following words have the following meaning associated therewith:
Analyte--the substance in a solution which is being analyzed, its concentration is unknown and is to be determined by the assay procedure. PA0 Reagent--a substance specifically chosen to react only with the analyte in the assay procedure. PA0 Label--a composition of the reagent, attached thereto, allowing for its detection by a measuring means. PA0 Distribution of label--the classification of the reagent into reacted and unreacted portions. PA0 Segregation of label--the classification made to occur between a solid and a liquid phase usually by chemical means. PA0 Suspension--a mixture of particles (solid phase) and liquid. PA0 Separation of label--the actual separation of particles and liquid by physical means.
Distribution of a reagent, following its reaction between particles and liquid in a suspension, is common in clinical chemistry. Examples of such distributions occur in radio-immuno assay and fluoro-immuno assay procedures. In both of these procedures, the concentration of an analyte in solution must be determined. A reagent, having a label attached thereto, is chosen to react specifically with the analyte. Following the reaction of the reagent with the solution, a portion of the reagent remains unreacted, while another portion of the reagent has reacted with the analyte. The reacted reagent is then segregated from the unreacted reagent causing a distribution thereof, usually by causing one of the reagents (reacted or unreacted) to enter into the solid phase, while the other remains in liquid phase. For example, this may occur by adsorbing either the reacted or unreacted reagent on a solid phase substance such as antibody coated beads, or charcoal, or precipitating the reacted reagent by the addition of precipitate causing substances such as polyethyleneglycol or second antibodies. A suspension of reacted and unreacted reagents then occurs. This suspension must be physically separated into the solid phase and the liquid phase causing a separation of the reacted reagent portion from the unreacted portion. Once the separation is made, then the measurement of each separate portion can be made. The label which is associated with the reagent is typically a radioactive isotope in the case of radio-immuno assay and is a fluorescent material in the case of fluoro-immuno assay. Other labels may also be used. The reagent is chosen such that its reaction and subsequent distribution is effected only by a specific analyte whose concentration is being determined. The reagent has the label attached thereto in order that its distribution may be measured, usually by emission sensing means. The portion of the reagent which distributes as determined by measuring its label, is indicative of the quantitative measure of the analyte concentration being determined by the assay procedure. In both the radio-immuno assay and fluoro-immuno assay procedures, the reacted and unreacted portions of the reagent are made to separate between two phases, either of which can be measured after they, in turn, are separated by physical means. The particle portion or the liquid portion of the suspension is analyzed to determine the quantitative measure of the label that is contained therein. In this manner, the unknown concentration, i.e., the quantitative measure of the specific analyte may be calculated. In the case of radio-immuno assay procedure, the separate particles or liquid is analyzed by converting the nuclear radiation emitted by the label into visible light by a scintillation crystal. The visible light is then detected by a photomultiplier tube. In the case of fluoro-immuno assay procedure, the separated particles or the liquid is irradiated with a beam of electromagnetic radiation, typically UV light. The amount of label present within the separated particles or the separated liquid would then fluoresce. The amount of fluoresence is then detected by a photomultiplier tube. Thus, in the case of an immuno-assay procedure, there is a reaction involving a reagent having a label, with a subsequent distribution and segregation of the reacted and unreacted portions of the reagent into solid and liquid phases. The separation and washing of these phases require intensive human handling and are a source of imprecision and error in the assay procedure.
MIA, or Microscopical Image Analysis, involves the examination of a suspension containing particles of interest in a microscope. Images of the suspension under the microscope are taken and are digitized and processed by imaging techniques in order to extract information relating to the selected components of the image which are of interest. Such an apparatus has been proposed for use to analyze particles in a suspension such as blood. U.S. patent application Ser. No. 146,064 filed on May 2, 1980 in the names of Gunner Bolz and Sherman DeForest now U.S. Pat. No. 4,338,024 and assigned to the assignee of the present application, deals with one such MIA apparatus.